Tuberculosis (TB) continues to be a leading public health problem worldwide. Approximately 5-20% of TB patients also develop extrapulmonary tuberculosis (EPTB), which complicates diagnosis, has high mortality rate, and is difficult to treat. Our current knowledge regarding the mechanisms by which mycobacteria invade and spread into extrapulmonary tissues is incomplete. Research on EPTB using animal models is hampered by slow growth of M. tuberculosis on agar plates. Non-invasive in vivo optical imaging can complement classical anatomopathological studies, helping to unravel the intricacies of EPTB, especially the temporal aspects of invasion and initial pulmonary bacterial numbers. We have developed an in vivo imaging system, designated beta-lactamase reporter enzyme fluorescence (REF), for real-time imaging of M. tuberculosis pulmonary infection of live mice. It is based on near infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme naturally expressed by tubercle bacilli but not by their eukaryotic hosts. Substrates are composed of a NIR dye connected to a quencher through the beta-lactam ring. Once the beta-lactam ring is hydrolyzed by M. tuberculosis beta-lactamase, BlaC, the fluorescent dye is freed from the quencher, and produces fluorescence. We propose that REF can be used to study the temporal kinetics of EPTB and specific organ- involvement in live animals and to investigate bacterial genes and host factors involved. We will investigate dispositions and kinetics of two substrates for REF imaging in lungs and extrapulmonary organs in uninfected and infected mice;examine threshold of each substrate for detection of bacteria in mouse organs, especially extrapulmonary organs;evaluate the correlation between bacterial colony forming units (CFU) and fluorescent signal;and investigate the temporal sequence of EPTB development in various organs. To validate REF in investigating bacterial genes and host factors involved in EPTB, we will select one bacterial gene, hbhA, and one host gene, IFN-g, that have known involved in EPTB development. Mice infected with hbhA mutant strain, complement strain and wild parental strain will be imaged. The quantified fluorescent level and CFU will be compared among groups. For IFN-g, we will image infected IFN-g knock-out mice and wild parental mice, and compare the fluorescent levels. Both experiments will have CFU collected to validate imaging results. The success of this proposed study could facilitate tuberculosis research progress by allowing investigators to directly monitor M. tuberculosis extrapulmonary infection and quantify bacterial viability in live animals. This study will obtain the first temporal understanding of the process ad an organ-specific sequence of events in a live animal model. This system will allow elegant strategies for investigation of bacterial genes and host factors that play important roles in EPTB development. Identifying bacterial and host factors affecting EPTB would help in the development of improved diagnostic tools, anti-tuberculosis therapies and vaccines.

Public Health Relevance

Extrapulmonary tuberculosis complicates diagnosis, is hard to treat, and has high rate of mortality. We propose application of a non-invasive optical imaging method to investigating extrapulmonary tuberculosis pathogenesis in live animals. Success of this study will greatly facilitate tuberculosis research and help in development of diagnostic tools, anti-tuberculosis therapies and vaccines.